This invention relates to the field of vaccines, and in particular, methods for the production of modified forms of pneumolysin and their use in producing compositions for the immunization of mammals against infections caused by bacteria including Streptococcus pneumoniae. 
Streptococcus pneumoniae is the major cause of bacterial pneumonia, bacteremia, meningitis, and otitis media (Baltimore et al. in Bacterial infections of humans: Epidemiology and control Evans and Brachman eds, Plenum Press, New York, 1989 pp.525-546; Schuchat et al. N. Engl. J. Med. 1997, 337, 970-976). Even with appropriate antibiotic therapy, pneumococcal infections have been estimated to result in as many as 40,000 deaths a year in the United States (Fedson et al. Archives of Internal Medicine 1994, 154, 2531-2535; Fiebach et al. Archives of Internal Medicine 1994, 154, 2545-2557). In addition, pneumococci have gained increased resistance to penicillin and other antibiotics making the development of an effective vaccine to prevent pneumococcal infections a public health priority (Farr et al. Archives of Internal Medicine 1995, 155, 2336-2340). Since the current 23-valent pneumococcal capsular polysaccharide vaccine is ineffective in children less than two years old (Douglas et al. J Infect Dis 1983, 148, 131-137; Leinonen et al. Pediatric Infectious Disease Journal 1986, 5, 39-44), numerous groups are developing multivalent conjugate vaccines to prevent otitis media, the major indication in this age group.
Pneumolysin (PLY), a sulfydryl-activated cytolytic toxin, is produced by all types of Streptococcus pneumoniae (Kanclerski et al. J Clin Microbiol 1987, 25, 222-225) and is considered a major virulence factor in pneumococcal infection (Boulnois Journal of General Microbiology 1992, 138, 249-259). Genetically engineered PLY-negative mutant strains of S. pneumoniae have been shown to be significantly less virulent in mice (Berry et al. Microb Pathog 1992, 12, 87-93; Berry et al. Infection and Immunity 1989, 57, 2037-2042). Cytotoxicity of PLY to pulmonary endothelial and epithelial cells is well demonstrated in vitro (Rubins et al. Infection and Immunity 1992, 60, 1740-1746). In addition, PLY may be the principal cause of hearing loss and cochlear damage in a guinea pig model of pneumococcal meningitis (Winter et al. Infection and Immunity 1997, 65, 4411-4418).
As of 1985, an estimated five million children under the age of 5 died from pneumonia caused by S. pneumoniae in developing countries each year. Lancet (1985) Sep 28 2(8457):699-701. S. pneumoniae employs a number of virulence factors to establish an initial infection and then produce invasive disease(s). To prevent systemic infections caused by the various serotypes of S. pneumoniae, immunization of infants and adults with suitable, cross-reactive vaccines, capable of eliciting safe, effective, and long-lasting immunity, is needed.
In a prospective study of pneumococcal colonization and infection in children, it was reported that pneumococcal serotypes 6, 14, 19, and 23 are the most commonly carried as well as the most frequent cause of infection in infants, mainly otitis media (Gray et al. J. Infect. Dis., 1988, 158, 948-955). In addition, it was recently found that these same strains are more frequent among the penicillin resistant clinical isolates (Nesin et al. J. Infect. Dis., 1998, 177, 707-713). Clinical studies carried out in young infants with a tetravalent pneumococcal conjugate vaccine including the above types, report a reduction in the carriage of vaccine-related strains (Dagan et al. Infect. Dis. J., 1997, 16, 1060-1064).
Almost all isolates of S. pneumoniae exhibit an external capsule made up of repeating oligosaccharides. Antigenic differences in the capsular polysaccharides due to different saccharide sequences are the hallmark of the different S. pneumoniae serotypes. Serotype-specific capsular polysaccharides are the major contributors to the virulence of the pneumococcus. Existing anti-pneumococcal vaccines are formulated from 23 capsular polysaccharides selected from the 84 serologically distinct types currently recognized. Unfortunately, these vaccines are not effective in all populations, especially those of Asia. A second shortcoming of the current vaccines is that polysaccharides by themselves are poor immunogens, especially for infants and the elderly.
Polypeptides expressed by S. pneumoniae also play an important pathogenic role. Some of the defined polypeptides that appear to contribute to the virulence of this organism include pneumolysin, autolysin, neuraminidase, pneumococcal surface polypeptide A (PspA), the 37 kDa polypeptide, adhesion molecules, hyaluronidase, and an IgA1 protease.
Virtually all serotypes of S. pneumoniae produce pneumolysin, one of the major virulence factors. This expression by the various S. pneumoniae serotypes makes pneumolysin a prime candidate for use in a protective vaccine against pneumococcal infections provided its toxicity can be altered.
Pneumolysin is an intracellular bacterial polypeptide with a molecular weight of approximately 53-kD. (Kanclerski et al. (1987) J. Clin. Microbiol. 25:222-225.) It is a member of a family of thiol-activated hemolysins and has various effects on eukaryotic cells. Pneumolysin is known to bind to cholesterol molecules in the eukaryotic membrane, form oligomers, and generate transmembrane pores. It has also been demonstrated that the respiratory burst, chemotactic, and phagocytic functions of polymorphonuclear leukocytes, all of which are critically important for removing invading pneumococci, are severely compromised in the presence of pneumolysin.
Pneumolysin causes both cytolytic and cytotoxic effects, and can stimulate an inflammatory response by the complement activation pathway. Nonspecific activation of complement causes depletion of complement polypeptides and generates nonspecific inflammation. Inoculation of pneumolysin into lungs of experimental animals causes pneumoniae-like symptoms. However, pre-immunization with pneumolysin is protective for experimental animals upon challenge with pneumococci. Paton et al. (1983) Infect. Immun. 40:548-552.
Because of pneumolysin""s immunogenic activity and capacity to elicit a protective response in individuals immunized with it, it has been suggested to use pneumolysin as a component of a vaccine. See PCT/AU89/00539. However, before pneumolysin can be included in vaccines for human use, this toxin must be modified so as to be substantially non-toxic while retaining the capacity to elicit protective antibodies.
Modified pneumolysins devoid of toxic activities are reported to have been generated based on the identification of amino acid regions of pneumolysin thought to have similar functions to related thiol-containing polypeptides. (WO 90/06951). The reported mutations are exclusively in the C-terminal portion of the polypeptide and were generated using targeted mutagenesis techniques. Other mutations, including certain specific amino acids in the N-terminal region have been reported to reduce hemolytic activity. The most significant reduction in hemolytic activity is reported as possibly being a result of histidine modification at position 156. Hill et al. (1994) Infection and Immunity, 62, 757-758. No data is provided concerning whether any of these substituted pneumolysins were properly refolded. A single mutation, Thr-172xe2x86x92Ile was reported to be responsible for a pneumolysin with reduced hemolytic activity. However, anomalous electrophoretic mobility indicates that the protein is incorrectly folded. Lock et al. Microb. Pathog. (1996) 21, 71-83.
This invention provides a novel method for generating and identifying stable, genetically modified, substantially non-toxic, immunogenic pneumolysin polypeptides using random PCR mutagenesis. Modified pneumolysin (pneumolysoid) which can be used as immunogens in a vaccine or can be used as an immunogenic carrier polypeptide for polysaccharide conjugate vaccines against S. pneumoniae or other bacterial infections are also provided. The modified pneumolysin polypeptides of this invention, while exhibiting substantially reduced or none of the toxin""s toxic activity, elicit antibodies which are cross-reactive with those elicited by the native toxin.
This invention also relates to nucleic acid sequences encoding the modified pneumolysins, vectors containing them as well as transformed host cells capable of expressing the nucleic acid molecules of this invention.
Another embodiment of this invention is polysaccharide-polypeptide conjugate molecules in which the modified pneumolysin of this invention is covalently coupled to bacterial polysaccharide to form the conjugate. Such conjugate molecules are useful as immunogens for eliciting a T cell dependent immunogenic response directed against the bacterial polysaccharide conjugated to the modified pneumolysin.
The invention is further directed to pharmaceutical compositions containing the modified pneumolysin polypeptides of the invention which elicit an immune response.
This invention further relates to a method of eliciting the production of antibodies reactive to the modified pneumolysin polypeptides. Such antibodies may be used to elicit both active and passive immunity. The modified pneumolysins of this invention may also be used to identify and isolate reactive antibodies.
It is therefore an object of this invention to provide genetically stable, modified S. pneumoniae pneumolysin polypeptides which have substantially attenuated or absent toxicity while retaining epitopes which cause production of antibodies which also bind the native toxin molecule.
It is a further object of this invention to provide a method for generating genetically modified pneumolysins(pneumolysoids).
It is another object of this invention to provide vaccine preparations comprising a modified pneumolysin polypeptide that can elicit antibodies and induce protective immunity against Streptococcus pneumoniae when delivered to a susceptible mammal. Such vaccines may be based on the pneumolysoid itself, or conjugates that comprise one or more bacterial polysaccharides covalently bound to a modified pneumolysin polypeptide of this invention.